Tip60 Might Be a Candidate For The Acetylation of Hepatic I and III in Mice

Nurdan GONUL BALTACI Ataturk University: Ataturk Universitesi Enver Fehim KOCPINAR Muş Alparslan Üniversitesi: Mus Alparslan Universitesi Harun Budak (  [email protected] ) Ataturk University https://orcid.org/0000-0002-7371-8959

Research Article

Keywords: Carbonic anhydrases, Tip60, acetylation, circadian rhythm, mice

Posted Date: March 22nd, 2021

DOI: https://doi.org/10.21203/rs.3.rs-323673/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/15 Abstract

Carbonic anhydrases (CAs) play an important role in maintaining pH balance by catalyzing the conversion of to . Since this pH balance is critical to health, all organisms must develop mechanisms to control and regulate it. Although there is a great deal of literature on the biochemical, functional, and structural properties of the CA family, there is no enough knowledge on the regulation of CAs at and protein levels, especially their epigenetic regulation. In this study, impact of Tip60, a member of histone acetyltransferases family, on the expression of Ca1 and Ca3 in liver tissue was investigated at different zeitgeber time points in control and liver-specifc Tip60 knockout mice (mutant) groups. First of all, Tip60 was specifcally knocked out in mouse liver the using Cre/loxP system and knockout rate was shown as 83% - 88% by southern blot. Expression profles of Ca1 and Ca3 genes in both groups were determined by Real-Time PCR at six different time points. While Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profle was observed at ZT0 and ZT20. Hepatic Ca1 showed a robust circadian expression. While hepatic Ca3 showed almost the same level of expression at different time units. The expression of Ca1 and Ca3 signifcantly decreased in the absence of Tip60 in mouse liver all time period. In conclusion, it was suggested for the frst time that Tip60 may be considered a candidate protein in the regulation of Ca1 and Ca3 genes, possibly by acetylation.

Introduction

Carbonic anhydrases (CAs), zinc-containing metalloenzymes that are commonly found in living organism, catalyze the reversible hydration of CO2 to bicarbonate (HCO-3) and proton (H+) [1-3]. Although these were frst discovered in hemolyzed blood, today it is known to have activity in many tissues such as kidney, liver, brain, muscle, and bone tissues [4-7]. CAs found in vertebrates (α-CA) are composed of 16 different isoenzymes and play a role in a wide variety of functions such as respiration, acid-base homeostasis, ion transport, bone resorption, taste preferences, ureagenesis, and gluconeogenesis [8-10]. Eight of these proteins are in the (CAI, CAII, CAIII, CAVII, CA VIII, CA X, CA XI and CA XIII), fve are transmembrane or membrane bound (CAIV, IX, XII, XIV and XV), two are in mitochondria (CAVA and VB), and one (CAVI) is secreted [11]. While there is a great deal of literature on the biochemical, functional, and structural properties of the CA family, there is no enough knowledge on the regulation of CAs at gene and protein levels, especially their epigenetic regulation [12]. Epigenetic mechanisms such as acetylation, phosphorylation, methylation, ubiquitination, sumoylation, and glycosylation are required to regulate and chromatin structure in mammalian cells without modulating the DNA sequence [13, 14]. Protein acetylation, which refers to the covalent binding of an acetyl group to an amino acid residue of a protein, is the most well-known along with phosphorylation [15]. The correlation between increased transcription and histone acetylation has been known for many years. Thus, acetylation regulates a number of metabolic and physiological processes by affecting protein functions, protein-DNA and protein-protein interactions, and subcellular localization of the protein [16, 17]. A recent study reported that members of the α-CA family, CAI (at the N-terminus), CAII (at the N-terminus, K18, K39, and K113), CAIII (at the N-terminus and K126), and CAXII (at K194), are acetylated by acetyltransferase . But

Page 2/15 the proteins involved in this acetylation are still unknown [12]. TIP60 protein, a member of the histone acetyltransferases (HATs) protein family, has important and vital functions such as transcriptional regulations, DNA repair, cell cycle, mechanism, cancer, circadian system and generation of cellular signals, both directly and indirectly. [18-21]. Studies have shown that the Tip60 protein is associated with many transcription factors and proto-oncogenes such as androgen receptor, c-Myb, c- Myc, STAT3, NF-Kb, E2F1, p53 and acts as a regulator / correlator [22-24]. The study was performed by Chen et al. showed that c-Myb transcription factor increases Ca1 expression by binding to its promoter in mouse erythroleukemia cells [25]. However, c-Myb inactivation is required to inhibit the Ca1 gene when the division of the cell is achieved. The cell goes to cancer without this suppression. It is also known that Tip60 is a regulating factor for c-Myb [22]. In this study, it is aimed that does Tip60 have a role in the regulation of hepatic Ca1 and Ca3 which are predominantly expressed in the liver? For this purpose, liver- specifc Tip60 knockout mice was generated by using Cre/loxP recombination. Quantitative expression of Ca1 and Ca3 genes at different zeitgeber time (ZT) points were determined for both control and knockout groups and then compared each other. Ca7, which is not regulated by acetylation, was used as a negative control [12]

Materials And Methods

2.1. Liver-Specifc Conditional Knockout Mouse Model

To generate liver-specifc Tip60 knockout mice (mutant), Tip60 foxed mice (10-12 week old male) with loxP sites fanking exons 1 and 9 of the Tip60 gene [21] were crossed to a SACre driver mouse line resulting in Cre-mediated deletion of Tip60 in the liver [26]. Mice were previously backcrossed to a C57BL/6N background for at least 10 generations. To delete Tip60, 10-12 week-old male mice (Tip60f/- ; SA+/Cre-ERT2) were injected daily with tamoxifen (10mg/ml stock solution, Sigma, St. Louis, MO, USA) in corn oil for fve consecutive days. The control group (Tip60f/f;SA+/+), in which corn oil were injected. Genotyping was performed with gene-specifc primers [21, 26]. Liver and kidney tissues were collected fve days after the last injection. Genomic DNA was isolated from both tissues and analyzed by southern blot.

2.2. Southern Blot Analysis

Genomic DNA from liver and kidney tissues was isolated with the DNeasy Tissue kit (Qiagen Inc., Valencia, CA, USA) and digested with BamHI (NEB, Ipswich, MA, USA). DNA was separated on a 0.6% agarose gel and transferred onto Hybond-XL positive charged nylon membrane (GE Healthcare/Amersham Biosciences, Sweden). The membranes were hybridized with a 32P-dCTP labeled radioactive double-stranded DNA probe was prepared by random priming using an appropriate commercial kit according to manufacturer’s instructions (Amersham Rediprime™ II DNA Labeling System, GE Healthcare) and purifed with the illustra ProbeQuant™ G-50 Micro Columns (GE Healthcare). Hybridization of the radioactive probe (100 µl) to the membrane was performed at 65°C overnight in the presence of a hybridization buffer. Membranes were washed with 2xSSC/0.1% SDS, 1xSSC/0.1% SDS,

Page 3/15 and 0.1xSSC/0.1% SDS, at 60°C until the excess label was removed and exposed to a sensitive X-ray flm (Kodak X-Omat 1000,1000A ve 1000J Processors).

2.3. Experimental Animals, Feeding, and Zeitgeber Time

At least 3 weeks prior any experiment, all mice were singly housed with food and water ad libitum under a 12‐hour‐light/12‐hour‐dark cycle (350 lux). Throughout this study, time is indicated using zeitgeber time (ZT) as the indicator for the phase of the rhythm, wherein ZT0 refers to the time that lights went on (06:00), and ZT12 refers to the time that the lights went off (18:00). ZT4, ZT8, ZT16, and ZT20 in this study are equivalent to 10:00, 14:00, 22:00, and 02:00 respectively [27]. Artifcial light was provided daily from ZT0 (06:00), with temperature (24 ± 1) °C, and humidity (55 ± 5%) kept constant [28]. In the frst set of experiments, 10–12‐week‐old male C57BL/6N mice were used and split up into six groups corresponding to the six chosen timepoints (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20). For the second set of experiments, we used 10–12‐week‐old male mutant mice (Tip60f/- ; SA+/Cre-ERT2) and their respective control littermates [29].

2.4. RNA Extraction and First Strand cDNA Synthesis

Total RNA isolation from approximately 50 mg mice liver tissues was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen-74804) following the manufacturer’s instructions. Concentrations and purities of RNAs were measured by spectrophotometer. (Thermo Scientifc, Multiskan GO, USA), RNA quality was checked by agarose gel electrophoresis and stored at −80°C until use. Total RNA was converted into frst strand cDNA using SuperScript III First-Strand cDNA kit system (Invitrogen, California, USA), utilizing random hexamers, according to the manufacturer's protocol. The resulting cDNAs were diluted to 100 ng/ μL with nuclease-free water and stored at −20 °C [30].

2.5. Primers and Probes Design

Primer3 software (v. 0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/) was used for gene-specifc primers and probes design that met the following criteria; amplicon size 75-200 bp, ≤3 G or C repetitions, GC content 50-65%, ≤4 base repetitions, melting temperature (Tm) 60°C. Primers and probes were verifed with Blast Tool (NCBI) to confrm its specifcity for the desired target. Then they were synthetized and purchased from Methabion International (Martinsried, Germany). Gene symbols and GenBank ID numbers are; Ca1 (Gene ID: 12346), Ca3 (Gene ID: 12350), Ca7 (Gene ID: 12354), and Actb (Gene ID: 11461). Since housekeeping genes were not affected by any of treatments, β-actin was used as reference gene. The sequences of specifc primers of all genes were shown in Table 1.

2.6. Quantitative Real‐Time PCR

To determine expression levels of Ca 1, Ca 3, and Ca 7 genes in different circadien points, Real-time PCR (qPCR) was carried out on Rotor-Gene Q instrument (QIAGEN, Inc., Hilden, Germany). Beta-actin was selected as reference control gene. The qPCR reactions were carried out with 2 µL of cDNA (fnal

Page 4/15 concentration is 0.02ng), 4 pmol of TaqMan probe, 8 pmol of forward and reverse primers and 10 µL FastStart TaqMan Probe Master Mix (Roche Diagnostics GmbHCorp, Mannheim, Germany) in a fnal volume of 20 µL. Optimal cycling conditions were 50 °C for 2 min, 95 °C for 10 min, 45 cycles of 95 °C for 15s, and annealing and extension at 60 °C for 1 min [31]. The expression results were analyzed using the ΔCT method [32].

2.7. Statistical Analysis

Each group contained three animals, and all measurements were triplicated for each animal. Statistical analysis was performed for each experiment using one‐way and two way analysis of variance (ANOVA) with Tukey's and Bonferroni's multiple comparisons test using the Prism software 7.0 (GraphPad Software, San Diego, CA). A value of P < .05 was considered to indicate a statistically signifcant difference (*P < .05, **P < .01, ***P < .001, **** P < .0001).

Results

3.1. Liver Specifc Tip60 Knockout Mice Models

Tamoxifen-inducible Cre/lox recombination was used to perform genetically modifed mouse lines in C57BL/6N backgrounds tissue-specifc or conditional knockouts of Tip60 due to the lethal effect of full knockout on mice. Tip60 gene was specifcally knocked out in mouse liver the using this system and the knockout rate was shown as 83% - 88% by southern blot. The leakage of CRE driver also checked in kidney tissues and was not seen any leak within the kidney tissues (Fig. 1).

3.2. Expression levels of Ca1, Ca3, and Ca7 genes at different zeitgeber time periods in the control groups

Circadian expression of Ca1, Ca3, and Ca7, which are expressed in the liver, was investigated in mice at six different ZT points in the contol group. While Ca1 showed the highest expression at ZT8 and ZT12, the lowest expression profle was observed at ZT0 and ZT20. Hepatic Ca1 showed a robust circadian expression. While hepatic Ca3 showed almost the same level of expression at different ZT periods, it revealed no circadian expression. While the highest expression of Ca7 was observed at ZT12, the lowest expression was seen at ZT4. Furthermore, hepatic Ca7 was also showed a robust circadian expression (Table 2).

3.3. Comparison of gene expression levels of control and mutant groups

A recently reported that while CAI and CAIII are acetylated by acetyltransferase enzymes, CAVII is not acetylated [12]. However, the proteins involved in this acetylation are still unknown. Since Tip60 is thought to be a candidate for the acetylation of Ca1 and Ca3 in the liver, quantitative expression levels of Ca1 and Ca3 in control and mutant groups were analyzed by qPCR. As a result of the analysis, it was determined that the expression amount in mutant tissues decreased signifcantly in all time points compared to control groups. No change was observed in the expression of Ca7 used as negative control. (Figure 2). Page 5/15 Discussion

CO2 is one of the simplest molecules involved in important physiological processes for all life domains. It is produced as part of the metabolic process and is quickly transported in the body through the blood [33,

34]. The acidity of the blood increases due to the high solubility and rapid spread of CO2. Since the pH of the human body is critical to health, all organisms must develop mechanisms to control it [35, 36]. CAs − are an important family of enzymes that catalyze the conversion of CO2 into HCO3 to regulate the pH balance of the blood. [37-39].

Many studies have proved the important role of CAs in physiological processes and showed that abnormal activity levels of these enzymes are associated with various human diseases such as glaucoma, erythroleukemia malignant brain tumors, and renal, gastric, and pancreatic carcinomas [35, − 40]. Furthermore, HCO3 produced by carbonic anhydrases is essential for the function of metabolic liver enzymes that performs many functions, including digestion, glycogen synthesis, manufacturing triglycerides and cholesterol, bile production, storage for many essential vitamins and minerals [41]. It is also metabolizing many drugs, medications, chemicals, and natural substances. Although several CA isoforms, including CA I, CA II, CA III, CAVII and CA IX, have been described in liver, there is very limited information about regulation of CAs at the gene and protein levels, especially their transcriptional regulation [4, 42-44].

As a result of the analysis, Ca1, Ca3, and Ca7 genes were expressed all ZT points in the liver tissues of control group. It was observed that the expression of Ca1 and Ca7 reached the maximum level in ZT8 (end of light cycle) and ZT12 (beginning of dark cycle) time unit and Ca3 gene was expressed high amounts in all time units except ZT16. In control group, comparing quantitative expression levels of Ca1, Ca3, and Ca7 in liver tissues, genes with the highest expression were Ca3, Ca1, and Ca7 respectively. In this respect, our fndings are consistent with the ‘’NCBI-Mouse ENCODE transcriptome data’’[45].

In the study performed by Chen et al in 2006 in mouse erythroleukemia (MEL) cells that c-Myb transcription factor binds to the promoter of carbonic anhydrase 1 (Ca1) gene and increases the expression of Ca1 for proliferation and differentiation [25]. Zhao et al. was stated that TIP60, a histone acetyl transferase with activity in cytoplasm and nucleus, belongs to MYST (Moz-Ybf2 / Sas3-Sas2- Tip60) family and is known to be responsible for acetylation in both mouse and human cells, is a regulating factor for c-Myb [22, 46-48]. In another study, the relationship between hepatocellular carcinoma (HCC) and tumor development with the expression of CAs was studied. The activity and protein expression of CA family in tumor tissues were observed to be signifcantly lower than normal cells [49]. Recent study reported by Di Fiore A, et al., CA I and CA III proteins are regulated by post-translational acetylation [12].

Many researchers have reported that knockout mice are widely used to better study the biological role of specifc genes, as well as molecular and cellular mechanisms [50-52]. The study by Hu et al. showed that homozygous disruption of the Tip60 gene lead to early embryonic death [53]. Therefore, we generated

Page 6/15 liver-specifc Tip60 knockout mice using the tamoxifen-inducible Cre/ loxP system to investigate the role of Tip60 in the regulation of Ca1 and Ca3 was investigated at the gene level. In addition, since TIP60 has been shown to have a role in the regulation of the circadian clock [21], it has been investigated whether this arrangement occurs in different ZT points. As shown in fgure 1, Tip60 gene knockout rate was shown between 83% - 88% by southern blot technique in the liver. The leakage of CRE driver also checked in kidney tissues and was not seen any leak within the kidney tissues.

The knockout rate of Tip60 in the liver tissue obtained from our study is efcient and useful for further studies as shown in the literature [54, 55]. And then, we investigated the expression of Ca1 and Ca3 genes at six different ZT points in the control and mutant groups. While the expression of Ca1 and Ca3 signifcantly decreased in the absence of Tip60 in mouse liver all time period (Figure 2a and b), the expression of Ca7, which is a negative contol as we mentioned above, was not affected (Figure 2c). Potter and Harris stated that some cytoplasmic CAs are markers for human cancers [56]. Bekku et al., reported that Ca1 expression decreased in colorectal tumors [57]. Chiang et al., observed that expression of Ca1 decreased in adenocarcinoma [58]. Kuo et al. revealed that reduced levels of CA I and III in human hepatocellular carcinoma (HCC). In contrast to this, in 2008, a study showed that increased CA III expression accelerates HCC through the focal adhesion kinase signaling pathway [59]. It was hypothesized that CA III is re-expressed in later stages of metastatic progression of HCC, and it might have an important infuence in the development of metastasis in liver cancer [39]. Following the results obtained from these two studies, It is hypothesized that while the decreased expression of Ca3 is important in the pathogenesis of HCC, increased Ca3 expression is required to metastasis after the process of cancer formation [39]. Based on the literature data, it is thought that decrease in Ca1 and Ca3 expression in mice due to the depletion of Tip60 may lead to HCC.

In conclusion, TIP60 may be considered a candidate protein in the regulation of Ca1 and Ca3 genes, possibly by acetylation. Moreover, our results show that Tip60 could be a new actor in explaining the molecular mechanism of hepatocellular carcinoma. However, it is clear that more studies including in vitro and in vivo tests are needed to support this hypothesis.

Declarations

Author contributions

Conceived and designed the experiments: HB (group leader). Performed the experiments: NGB, EFK, and HB. Analyzed the data: NGB, EFK, and HB. Contributed reagents/materials/analysis tools: HB. Wrote the paper: HB and NGB. All authors read and approved the fnal manuscript.

Acknowledgements

We thank Dr. Gregor Eichele at the Max Planck Institute (MPI) for Biophysical Chemistry, Germany for providing liver-specifc Tip60 knockout mice and Dr. Pierre Chambon at the Institute National de la Sante´ et de la Recherche Me´ dicale, Universite´ Louis Pasteur, France for providing SACre driver mouse line. The

Page 7/15 authors would like to thank Dr. M. Özkan Baltaci for reading the manuscript and providing useful suggestions. This work was funded by grants from Atatürk University Scientifc Research Projects Coordination Commission [Grant Numbers: PRJ2010/279].

Confict of interest

The authors declare that there is no potential confict of interest with respect to the research, authorship, and/or publication of this article. All authors read and approved the fnal manuscript.

Compliance with ethical standards

This article does not contain any studies with human participants. Animal experimentation: Mouse handling was carried out in accordance with the German Law on Animal Welfare and was ethically approved and licensed by the Ofce of Consumer Protection and Food Safety of the State of Lower Saxony (license numbers 33.11.42502-04/072/07 and 33.9-42502-04-12/0719).

References

1. Gilmour KM: Perspectives on carbonic anhydrase. Comp Biochem Physiol A Mol Integr Physiol 2010, 157(3):193-197. 2. Ozensoy Guler O, Capasso C, Supuran CT: A magnifcent enzyme superfamily: carbonic anhydrases, their purifcation and characterization. J Enzyme Inhib Med Chem 2016, 31(5):689-694. 3. Li Z, Jiang L, Toyokuni S: Role of carbonic anhydrases in ferroptosis-resistance. Arch Biochem Biophys 2020, 689:108440. 4. Cankaya M, Hernandez AM, Ciftci M, Beydemir S, Ozdemir H, Budak H, Gulcin I, Comakli V, Emircupani T, Ekinci D et al: An analysis of expression patterns of genes encoding proteins with catalytic activities. Bmc Genomics 2007, 8. 5. Haapasalo J, Nordfors K, Haapasalo H, Parkkila S: The Expression of Carbonic Anhydrases II, IX and XII in Brain Tumors. Cancers 2020, 12(7). 6. Shaikh AB, Fang HW, Li M, Chen SY, Shan P, Shang XL: Reduced expression of carbonic anhydrase III in skeletal muscles could be linked to muscle fatigue: A rat muscle fatigue model. J Orthop Transl 2020, 22:116-123. 7. Chang XT, Zheng YB, Yang QR, Wang L, Pan JH, Xia YF, Yan XF, Han JX: Carbonic anhydrase I (CA1) is involved in the process of bone formation and is susceptible to ankylosing spondylitis. Arthritis Res Ther 2012, 14(4). 8. Supuran CT: Carbonic anhydrase activators. Future Med Chem 2018, 10(5). 9. Li Z, Jiang L, Toyokuni S: Role of carbonic anhydrases in ferroptosis-resistance. Archives of Biochemistry and Biophysics 2020, 689. 10. Supuran CT: Diuretics: From classical carbonic anhydrase inhibitors to novel applications of the sulfonamides. Curr Pharm Design 2008, 14(7):641-648.

Page 8/15 11. Bayram E, Senturk M, Kufrevioglu OI, Supuran CT: In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase I and II. Bioorgan Med Chem 2008, 16(20):9101- 9105. 12. Di Fiore A, Supuran CT, Scaloni A, De Simone G: Human carbonic anhydrases and post-translational modifcations: a hidden world possibly affecting protein properties and functions. J Enzym Inhib Med Ch 2020, 35(1):1450-1461. 13. Chen JZ, Liu Q, Zeng LB, Huang XT: Protein Acetylation/Deacetylation: A Potential Strategy for Fungal Infection Control. Front Microbiol 2020, 11. 14. Li XJ, Egervari G, Wang YG, Berger SL, Lu ZM: Regulation of chromatin and gene expression by metabolic enzymes and metabolites. Nat Rev Mol Cell Bio 2018, 19(9):563-578. 15. Xia C, Tao Y, Li MS, Che TJ, Qu J: Protein acetylation and deacetylation: An important regulatory modifcation in gene transcription (Review). Exp Ther Med 2020, 20(4):2923-2940. 16. Kouzarides T: Acetylation: a regulatory modifcation to rival phosphorylation? EMBO J 2000, 19(6):1176-1179. 17. Narita T, Weinert BT, Choudhary C: Functions and mechanisms of non-histone protein acetylation. Nat Rev Mol Cell Biol 2019, 20(3):156-174. 18. Miyamoto N, Izumi H, Noguchi T, Nakajima Y, Ohmiya Y, Shiota M, Kidani A, Tawara A, Kohno K: Tip60 is regulated by circadian transcription factor clock and is involved in cisplatin resistance. J Biol Chem 2008, 283(26):18218-18226. 19. Sun Y, Jiang X, Price BD: Tip60: connecting chromatin to DNA damage signaling. Cell Cycle 2010, 9(5):930-936. 20. Sun Y, Jiang X, Chen S, Fernandes N, Price BD: A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM. Proc Natl Acad Sci U S A 2005, 102(37):13182-13187. 21. Petkau N, Budak H, Zhou X, Oster H, Eichele G: Acetylation of BMAL1 by TIP60 controls BRD4-P-TEFb recruitment to circadian promoters. Elife 2019, 8. 22. Zhao H, Jin S, Gewirtz AM: The histone acetyltransferase TIP60 interacts with c-Myb and inactivates its transcriptional activity in human leukemia. J Biol Chem 2012, 287(2):925-934. 23. Frank SR, Parisi T, Taubert S, Fernandez P, Fuchs M, Chan HM, Livingston DM, Amati B: MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO Rep 2003, 4(6):575-580. 24. Xiao H, Chung J, Kao HY, Yang YC: Tip60 is a co-repressor for STAT3. J Biol Chem 2003, 278(13):11197-11204. 25. Chen J, Kremer CS, Bender TP: The carbonic anhydrase I locus contains a c-Myb target promoter and modulates differentiation of murine erythroleukemia cells. Oncogene 2006, 25(19):2758-2772. 26. Schuler M, Dierich A, Chambon P, Metzger D: Efcient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse. Genesis 2004, 39(3):167-172. 27. Jiao X, Wu M, Lu D, Gu J, Li Z: Transcriptional Profling of Daily Patterns of mRNA Expression in the C57BL/6J Mouse Cornea. Curr Eye Res 2019, 44(10):1054-1066.

Page 9/15 28. Zhu X, Yang L, He Y, Sun Y, Shi W, Ou C: Liver Function of Male Rats Exposed to Manganese at Different Time Points. Biol Trace Elem Res 2020, 198(1):224-230. 29. Perreau-Lenz S, Zghoul T, de Fonseca FR, Spanagel R, Bilbao A: Circadian regulation of central ethanol sensitivity by the mPer2 gene. Addict Biol 2009, 14(3):253-259. 30. Budak H, Kocpinar EF, Gonul N, Ceylan H, Erol HS, Erdogan O: Stimulation of gene expression and activity of antioxidant related enzyme in Sprague Dawley rat kidney induced by long-term iron toxicity. Comp Biochem Physiol C Toxicol Pharmacol 2014, 166:44-50. 31. Ceylan H, Budak H, Kocpinar EF, Baltaci NG, Erdogan O: Examining the link between dose-dependent dietary iron intake and Alzheimer's disease through oxidative stress in the rat cortex. J Trace Elem Med Bio 2019, 56:198-206. 32. Pfaf MW: A new mathematical model for relative quantifcation in real-time RT-PCR. Nucleic Acids Res 2001, 29(9):e45. 33. Neri D, Supuran CT: Interfering with pH regulation in tumours as a therapeutic strategy. Nat Rev Drug Discov 2011, 10(10):767-777. 34. Supuran CT: Structure and function of carbonic anhydrases. Biochem J 2016, 473:2023-2032. 35. Alterio V, Di Fiore A, D'Ambrosio K, Supuran CT, De Simone G: Multiple Binding Modes of Inhibitors to Carbonic Anhydrases: How to Design Specifc Drugs Targeting 15 Different Isoforms? Chem Rev 2012, 112(8):4421-4468. 36. Capasso C, Supuran CT: An overview of the alpha-, beta- and gamma-carbonic anhydrases from Bacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria? J Enzym Inhib Med Ch 2015, 30(2):325-332. 37. Blandina P, Provensi G, Passsani MB, Capasso C, Supuran CT: Carbonic anhydrase modulation of emotional memory. Implications for the treatment of cognitive disorders. J Enzym Inhib Med Ch 2020, 35(1):1206-1214. 38. Lee D, Hong JH: The Fundamental Role of Bicarbonate Transporters and Associated Carbonic Anhydrase Enzymes in Maintaining Ion and pH Homeostasis in Non-Secretory Organs. Int J Mol Sci 2020, 21(1). 39. Mboge MY, Mahon BP, McKenna R, Frost SC: Carbonic Anhydrases: Role in pH Control and Cancer. Metabolites 2018, 8(1). 40. Clare BW, Supuran CT: A perspective on quantitative structure-activity relationships and carbonic anhydrase inhibitors. Expert Opin Drug Metab Toxicol 2006, 2(1):113-137. 41. Diez-Fernandez C, Rufenacht V, Santra S, Lund AM, Santer R, Lindner M, Tangeraas T, Unsinn C, de Lonlay P, Burlina A et al: Defective hepatic bicarbonate production due to carbonic anhydrase VA defciency leads to early-onset life-threatening metabolic crisis. Genet Med 2016, 18(10):991-1000. 42. Alchera E, Tacchini L, Imarisio C, Dal Ponte C, De Ponti C, Gammella E, Cairo G, Albano E, Carini R: Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells. Hepatology 2008, 48(1):230-239.

Page 10/15 43. Bejaoui M, Pantazi E, De Luca V, Panisello A, Folch-Puy E, Hotter G, Capasso C, Supuran CT, Rosello- Catafau J: Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage (vol 10, e0134499, 2015). Plos One 2015, 10(9). 44. Thiry A, Dogne JM, Masereel B, Supuran CT: Targeting tumor-associated carbonic anhydrase IX in cancer therapy. Trends Pharmacol Sci 2006, 27(11):566-573. 45. Yue F, Cheng Y, Breschi A, Vierstra J, Wu WS, Ryba T, Sandstrom R, Ma ZH, Davis C, Pope BD et al: A comparative encyclopedia of DNA elements in the mouse genome. Nature 2014, 515(7527):355-+. 46. Dong Y, Isono K, Ohbo K, Endo TA, Ohara O, Maekawa M, Toyama Y, Ito C, Toshimori K, Helin K et al: EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice. Molecular and Cellular Biology 2017, 37(19). 47. Li TY, Song LT, Sun Y, Li JY, Yi C, Lam SM, Xu DJ, Zhou LK, Li XT, Yang Y et al: Tip60-mediated lipin 1 acetylation and ER translocation determine triacylglycerol synthesis rate. Nat Commun 2018, 9. 48. Smith ER, Cayrou C, Huang R, Lane WS, Cote J, Lucchesi JC: A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16. Molecular and Cellular Biology 2005, 25(21):9175-9188. 49. Kuo WH, Chiang WL, Yang SF, Yeh KT, Yeh CM, Hsieh YS, Chu SC: The differential expression of cytosolic carbonic anhydrase in human hepatocellular carcinoma. Life Sci 2003, 73(17):2211-2223. 50. Dubois EL, Guitton-Sert L, Beliveau M, Parmar K, Chagraoui J, Vignard J, Pauty J, Caron MC, Coulombe Y, Buisson R et al: A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2. Nucleic Acids Res 2019, 47(14):7532-7547. 51. Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT et al: The knockout mouse project. Nat Genet 2004, 36(9):921-924. 52. Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T et al: A conditional knockout resource for the genome-wide study of mouse gene function. Nature 2011, 474(7351):337-U361. 53. Hu Y, Fisher JB, Koprowski S, McAllister D, Kim MS, Lough J: Homozygous disruption of the Tip60 gene causes early embryonic lethality. Dev Dyn 2009, 238(11):2912-2921. 54. Hanly PJ, Roberts D, Dobson K, Light RB: Effect of indomethacin on arterial oxygenation in critically ill patients with severe bacterial pneumonia. Lancet 1987, 1(8529):351-354. 55. Tang Y, Tang LY, Xu X, Li C, Deng C, Zhang YE: Generation of Smurf2 Conditional Knockout Mice. Int J Biol Sci 2018, 14(5):542-548. 56. Potter CP, Harris AL: Diagnostic, prognostic and therapeutic implications of carbonic anhydrases in cancer. Br J Cancer 2003, 89(1):2-7. 57. Bekku S, Mochizuki H, Yamamoto T, Ueno H, Takayama E, Tadakuma T: Expression of carbonic anhydrase I or II and correlation to clinical aspects of colorectal cancer. Hepatogastroenterology 2000, 47(34):998-1001.

Page 11/15 58. Chiang WL, Chu SC, Yang SS, Li MC, Lai JC, Yang SF, Chiou HL, Hsieh YS: The aberrant expression of cytosolic carbonic anhydrase and its clinical signifcance in human non-small cell lung cancer. Cancer Lett 2002, 188(1-2):199-205. 59. Dai HY, Hong CC, Liang SC, Yan MD, Lai GM, Cheng AL, Chuang SE: Carbonic Anhydrase III Promotes Transformation and Invasion Capability in Hepatoma Cells Through FAK Signaling Pathway. Mol Carcinogen 2008, 47(12):956-963.

Tables

Table 1. Sequence of PCR primers and TaqMan probes for genes used in qPCR.

Primer name Primer sequence (5′–3′) Accession Product Tm number Size (oC)

Ca1-Forward AGAGCCTGCAGTTCCAGTTC XM_011248137.1 97 bp 60

Ca1-Reverse CTCATTCCTTGCTGGGACTC

Ca1-HYB Oligo FAM-TGAGCAACCACCGTCCACCC- 70 TAMRA

Ca3-Forward ACACACTTTGACCCATCATG NM_007606.3 130 bp 60

Ca3-Reverse GAGCTCACAGTCATGGGCTC

Ca3-HYB Oligo FAM- TGTTCCCTGCTTGCCGGGAC- 70 TAMRA

Ca7-Forward TGGTTCACTGGAACGCCAAG NM_053070.3 143 bp 60

Ca7-Reverse AACCATGTAGAGGGCGTCTG

Ca7-HYB Oligo FAM-TGGCCTGGCTGTGGTTGGTG- 70 TAMRA

β-actin-Forward AATCGTGCGTGACATCAAAG BC138614.1 137 bp 60

β-actin-Reverse CGTTGCCAATAGTGATGACCT

β-actin HYB Cy5- ATGGCCACTGCCGCATCCTC- 70 Oligo BQ2

Table 2. Comparison of mRNA levels Ca1, Ca3 and Ca7 in liver tissues at different zeitgeber time points

Page 12/15 Groups Ca1 Ca3 Ca7

Difference p values Difference p Difference p values between between values between means means means

ZT0 & -0,00108 ± 0,9928ns 0,0614 ± 0,9998 0,0003 ± 0,1110 ns ZT4 0,00148 1,423 ns 8,386e-005

ZT0 & -0,00736 ± 0,0077** 0,1946 ± 0,9591 -0,0001867 ± 0,4711 ns ZT8 0,00162 0,148 ns 0,0001107

ZT0 & -0,00792 ± 0,0037** 0,1757 ± 0,9735 -0,0004517 ± 0,0017** ZT12 0,00232 0,247 ns 4,686e-005

ZT0 & -0,0009 ± 0,9976ns 0,1901 ± 0,9615 0,0001767 ± 0,5305 ns ZT16 0,00124 0,2412 ns 5,496e-005

ZT0 & 0,00018 ± >0,9999ns 0,28 ± 0,184 0,8347 3,9e-005 ± 0,9991 ns ZT20 0,00073 ns 0,0001205

ZT4 & -0,00628 ± 0,0306* 0,1332 ± 0,9923 -0,0004617 ± 0,0013** ZT8 0,00205 0,1786 ns 0,000128

ZT4 & -0,00684 ± 0,0153* 0,1144 ± 0,9962 -0,0007267 ± <0,0001**** ZT12 0,00263 0,2662 ns 7,961e-005

ZT4 & 0,00018 ± >0,9999ns 0,1287 ± 0,9930 -9,833e-005 0,9276 ns ZT16 0,00186 0,2609 ns ± 8,463e-005

ZT4 & 0,00126 ± 0,9856ns 0,2186 ± 0,9342 -0,000236 ± 0,2727 ns ZT20 0,00145 0,2092 ns 0,0001399

ZT8 & -0,00055 ± 0,9997ns -0,0188 ± >0,9999 -0,000265 ± 0,1348 ns ZT12 0,00272 0,2494 ns 0,0001075

ZT8 & 0,00646 ± 0,0355* -0,0045 ± >0,9999 0,0003633 ± 0,0158* ZT16 0,00201 0,2439 ns 0,0001113

ZT8 & 0,00754 ± 0,0061** 0,0854 ± 0,9991 -0,0002257 ± 0,3179 ns ZT20 0,0016 0,1875 ns 0,0001611

ZT12 & 0,00702 ± 0,0185* 0,0144 ± >0,9999 0,0006283 ± <0,0001**** ZT16 0,00271 0,3137 ns 4,824e-005

ZT12 & 0,0081 ± 0,0029** 0,1043 ± 2722 0,9976 0,0004907 ± 0,0011** ZT20 0,0023 ns 0,0001169

ZT16 & 0,00108 ± 0,9943ns -0,090 ± 0,9989 -0,0001377 ± 0,7944 ns ZT20 0,00121 0,2671 ns 0,0001212

Data are shown as the mean ± SEM. Difference between groups was evaluated with the unpaired Student t-test. Statistically signifcant differ-ences are indicated as follows: p > 0.05 (not signifcant, ns); *p < 0.05 (signifcant); **p < 0.01 (very signifcant); ***p < 0.001 (extremely signifcant), ****p < 0.0001 (extremely signifcant).

Page 13/15 Figures

Figure 1

Cre-mediated deletion of Tip60 in the mouse liver tissue showed by southern blot technique. Line1: Tip60f/-;SA+/+, Line2: Tip60f/-;SA+/Cre-ERT2, Line3: Tip60f/-;SA+/+, Line4: Tip60f/-;SA+/Cre-ERT2 (mutant). Densities and percentages of blot lines were measured quantitatively using the Image J 2.0 software (NIH, USA) (E).

Figure 2

Page 14/15 Comparison of genes expression levels in control and mutant tissues. Ca1, Ca3 and Ca7. Changes in the gene expression levels of Ca1 (a), Ca3 (b) and Ca7 (c) were detected by qPCR. β-actin was used as a housekeeping gene.

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